Synthesis, bioactivity assessment, molecular docking and ADMET studies of new chromone congeners exhibiting potent anticancer activity

In consideration of the chromones' therapeutic potential and anticancer activity, a new series of chromanone derivatives have been synthesized through a straightforward reaction between 6-formyl-7-hydroxy-5-methoxy-2-methylchromone (2) and various organic active compounds. The cytotoxic activity of the newly synthesized congeners was investigated against MCF-7 (human breast cancer), HCT-116 (colon cancer), HepG2 (liver cancer), and normal skin fibroblast cells (BJ1). The obtained data indicated that compounds 14b, 17, and 19 induce cytotoxic activity in the breast MCF7, while compounds 6a, 6b, 11 and 14c showed highly potent activity in the colon cancer cell lines. Overall, the results demonstrate that the potential cytotoxic effects of the studied compounds may be based on their ability to induce DNA fragmentation in cancer cell lines, down-regulate the expression level of CDK4 as well as the anti-apoptotic gene Bcl-2 and up-regulate the expression of the pro-apoptotic genes P53 and Bax. Furthermore, compounds 14b and 14c showed a dual mechanism of action by inducing apoptosis and cell cycle arrest. The docking studies showed that the binding affinity of the most active cytotoxic compounds within the active pocket of the CDK4 enzyme is stronger due to hydrophobic and H-bonding interactions. These results were found to be consistent with the experimental results.

Figure 1 displays the structures of some biologically active chromones.Compound I demonstrated potent cytotoxic activity against various cancer cell lines, including leukaemia, colon, prostate, and melanoma 15 .Chromone derivative II, in turn, induced apoptosis in both lung and breast cancer cell lines and showed selectivity for isoforms IX and XII of human carbonic anhydrase (hCA) 16 .Chromone attached to 1-alkyl-1H-imidazole-2-yl (III) demonstrated excellent activity against prostate cancer cell lines, and 4H-chromone-1,2,3,4-tetrahydropyrimdine-5-carboxylates (IV) displayed significant activity against leukaemia cell lines without showing toxicity to normal cell lines 8 .Additionally, Nam et al. developed chromone derivatives V and VI, which are analogues of lavendustin and both showed prominent activities against A-549 and HCT-15 cell lines 17 .
Because of their therapeutic behaviours and low toxicity, chromones are considered to be an attractive source for the development of new pharmaceuticals.In our ongoing efforts to develop novel chemopreventive treatments for cancer disorders and according to the fact that chromone moiety plays a significant role in the pharmacophores of numerous biologically active compounds with a variety of therapeutic uses [18][19][20][21][22] .The current study seeks to synthesize new chromone derivatives and evaluate their anticancer efficacy starting from the naturally occurring visnagin.

Result and discussion
Visnagin (1) was recognized as a "hit" (active) from the natural products repository screening in the search for new leads for curative cancer treatment.Additionally, it acts as a precursor in the synthesis of many bioactive compounds.Visnagin (1) was converted to 6-formyl-7-hydroxy-5-methoxy-2-methylchromone (2) by oxidation using 10% potassium dichromate 23 , which underwent further reactions with various organic reagents.The presence of both aldehydic and hydroxyl groups in 2 makes chemical modifications easier and more versatile.
A new starting compound ethyl 2-((6-formyl-5-methoxy-2-methyl-4-oxo-4H-chromen-7-yl)oxy)acetate (8) had been furnished through the reaction of 2 with ethyl bromoacetate and was used as a building block for many chroman-4-one derivatives.Compound 8 was prepared according to the reported method 24 and was obtained in high yield, in an extremely pure state and used without further purification (Fig. 2).

Evaluation of in vitro cytotoxic activity
The in vitro MTT assay was utilized to assess the cytotoxic activity of the newly synthesized chromone congeners 3-20 on various cancer cell lines including human breast cancer (MCF-7), human colon cancer (HCT-116), human liver cancer (HepG2) as well as human normal Skin fibroblast cells (BJ1).After 48 h of exposure to a single dose concentration of 100 μM, the percentage of cell death was computed in relation to the cells that were left untreated.

DNA fragmentation
The DNA fragmentation was examined in breast cancer cell lines (MCF-7) and colon cancer cell lines (HCT-116) using diphenylamine reaction procedure as well as by DNA gel electrophoresis laddering assay as described in the experimental section.
In the breast cancer cell lines (MCF-7), treatments with three different compounds 14b, 17, and 19 at IC50 concentration were investigated.The results demonstrated that the negative control cells had significantly lower (P < 0.01) DNA fragmentation rates than the treated groups.Compound 14b had the highest fragmentation rate, while compound 17 had the lowest as displayed in Table 3 and Fig. 5.  www.nature.com/scientificreports/On the other hand, in the colon cancer cell lines (HCT-116) four different treatments with compounds 6a, 6b, 11 and 14c were examined.The negative control had significantly lower (P < 0.01) DNA fragmentation rates compared to the treated groups.The positive control treated with Doxo showed the highest fragmentation rate and compounds 6a and 14c had higher fragmentation rates than the other tested compounds (Table 4, Fig. 6).www.nature.com/scientificreports/

Gene expression
The families of pro-and anti-apoptotic play a key factor in beating cancer growth their gene expression was assayed.

Effects of chromone congeners on mRNA expression of P53, BAX, BCL2 and CDK4 on breast cancer (MCF7)
The result indicated that the mRNA levels of P53 and Bax were up-regulated, while Bcl-2 and CDK4 were downregulated in breast cancer (MCF7) cell lines treated with compounds 14b, 17, 19 and DOX as compared to the untreated cells.Moreover, the effect of compounds 14b and 19 was significantly more potent than DOX (Fig. 7).

Effects of chromone congeners on mRNA expression of P53, BAX, BCL2 and CDK4 on colon cancer (HCT116)
The result revealed that treating HCT116 with compounds 6a, 6b, 11, 14c, and DOX resulted in increased mRNA levels of both P53 and Bax.However, these treatments also led to lower levels of CDK4 and Bcl-2 mRNA when compared to the negative control.Compounds 6a and 14c had significantly higher mRNA expression in P53 and Bax than DOX.Moreover, they also led to significant CDK4 and Bcl-2 down-regulation than DOX (Fig. 8).
On the other hand, the down-regulation of 11 was found to be similar to that of DOX as displayed in Fig. 8.
Recently, the inhibition of CDKs (cyclin-dependent kinases) that regulate the cell cycle, such as CDK4/6, is a crucial goal for cancer researchers to prevent inappropriate cell division and promote inhibitory barriers.In the transition of cells into the S phase, CDK4 and CDK6 play a critical role as mediators and are essential for the initiation, and survival of several cancer types 25 .
Our results indicate that all of the tested compounds were able to down-regulate CDK4 mRNA levels in treated cancer cells.It was observed that compounds 6a, 14b, 14c and 19 demonstrated a higher efficacy in comparison to DOX, whereas compounds 17 and 6b showed a lower efficacy than DOX.On the other hand, tumors generally show elevated Bcl-2 expression 26,27 .
The pro-apoptotic proteins in the Bcl-2 family, such as Bax and Bak, play an important role in promoting the release of cytochrome c and ROS, which are important signals in the apoptosis cascade.These pro-apoptotic proteins are activated by BH3-only proteins and inhibited by the function of Bcl-2 and its relative Bcl-Xl 28 .Therefore, down-regulating Bcl-2 and up-regulating Bax can be a hopeful approach to control tumorigenesis.www.nature.com/scientificreports/Our studied compounds were found to suppress Bcl-2 and activate Bax, which could contribute to the apoptosis mechanism.Furthermore, the up-regulation of P53 can also be vital for the activation of apoptosis 29 .It is known that P53 suppresses tumors by inhibiting cell proliferation through the activation of P21 protein, as well as by initiating apoptosis.The mechanism of P53's action is both transcriptionally dependent and independent 30 .Also, P53 plays a significant role in various cell signaling mechanisms, such as cell-cycle arrest, DNA repair, differentiation, and cell death (apoptosis).Thus, the ability of the tested compounds to up-regulate P53 can help activate apoptosis.It is worth mentioning that cancers in patients with changes or clampdown of P53 function are not responsive to conservative chemotherapeutic drugs, but rather respond to new genotoxic chemotherapeutics that act via the P53 pathway.

Flow cytometer analysis
Annexin V and propidium iodide staining are commonly used method for identifying apoptotic cellular death.In the presence of Ca 2+ ions, annexin V has a strong binding affinity for phosphatidylserine a membrane phospholipid that is translocated from the inner to the outer side of the cell membrane during apoptosis.However, propidium iodide has the ability to bind DNA can only enter into necrotic or late apoptotic cells.The effectiveness of various compounds on cancer cell lines can be ascertained using this technique for detecting apoptosis.

Effects of compounds on MCF cells
The apoptotic rate of compounds 14b, 17, 19 and DOX in the Breast cancer cell line (MCF7) was determined using the Annexin V-FITC/PI Double Staining Kit.
Compound 14b showed high significant increase in necrosis (16.37%) compared to the normal control (0.04%).In addition, early and late apoptosis were 2.08 and 2.78% respectively when compared with normal control (0.30%).Whereas, compounds 17 and 19, did not show any significant differences with normal control, but DOX showed highly increasing necrosis (99.83%) when compared with normal control (0.04%) Fig. 9.   www.nature.com/scientificreports/

Effects of compounds on HCT116 cells
The apoptotic rate of compounds 6a, 6b, 14c and DOX in colon cancer (HCT116) was determined using the Annexin V-FITC/PI Double Staining Kit.The results revealed that only compound 6b and DOX showed a highly significant increase in necrosis (31.69 and 99.53% respectively) when compared with normal control (0.02%).
While the apoptotic rate of 14c in colon cancer (HCT116) recorded no significant differences in necrotic cells (0.56%) when compared with normal control (0.02%) and the early apoptosis was significantly highly increased (9.98%) in these treated cells when compared with normal control (0.01%).Additionally, compounds 6a did not show significant necrosis in the cells (0.02%) when compared with negative control (0.02%) Fig. 10.

Effects of compounds 14b and 14c on cell cycle arrest
Furthermore, the impact of compounds 14b, and 14c on the cell cycle arrest and proliferation of both MCF7 and HCT116 cell lines were investigated after 24 h of treatment utilizing cellular DNA flow cytometry and the untreated cells were used as a negative control for comparison (Figs.11, 12).The results showed that in the MCF7 control group, the majority of cells were in the G0/G1 phase (81.94%), with only 5.84, and 0.81% in the S and G2-M phase, respectively.Upon treatment with compound 14b, there was a significant decrease in the G0/ G1 phase (32.53%), and a significant increase in the S phase (44.1%).Additionally, there was an increase in the G2-M phase (4.53%) in treated MCF7cells compared to the control (0.81%) Fig. 11.
On the other hand, the study conducted on the HCT116 cell line showed that in the negative control group, 73.64% of HCT116 cells were in the G0-G phase, whereas only 0.78% and 0.00% of cells in S and G2-M phases, respectively.However, upon treatment with compound 14c, the number of cells in the G0-G1 phase decreased significantly (56.18%) while the number of cells increased significantly in the S phase (24.43%) and G2-M phase (2.92%) as compared to the normal control (Fig. 12).
It is important to note that the G1 phase is the growth phase where everything is ready for DNA synthesis, while the S phase is the synthesis phase, and the G2 phase is the growth and preparation stage for mitosis.An agent that can arrest cell division in cancer cells in either the S or G2 phase is considered an anti-cancer agent.The potency of this agent is measured by the number of cells in the S and G2 phases, and there is an inverse relationship between the potency of anticancer activities and the number of cells in the S and G2 phases.Furthermore, cell cycle checkpoints in the S and G2 phases of the cell cycle are the major checkpoints and play an important role in cell cycle progress.Based on these findings, we hypothesize that both 14b and 14c have anticancer activities against MCF7 and HCT116 cancer cells, respectively.Additionally, our tested compounds upregulated the P53 expression, which causes cell cycle arrest via p21 activation 31 .Therefore, we propose that the resulting cell cycle arrest may be due to the increased expression of P53.

Molecular docking
The molecular docking technique was utilized to comprehend the binding interaction of the most active synthesized compounds 6a, 6b, 11, 14b, 14c, 17 and 19 with the binding sites of CDK4 (PDB ID:7SJ3).PyRx tools Autodock vina (version 8) were used to execute the docking technique.The lowest energy of binding (LEB) was calculated for each compound, which reflects the binding affinity of the compound.Additionally, hydrogen bonds and hydrophobic bonds such as carbon-hydrogen, van der Waals, Pi-sigma, alkyl, Pi-alkyl, etc. were evaluated 32 .To validate the docking protocol, the co-crystallized ligand was re-docked into the enzyme's active site, and it was found that the re-docked ligand overlapped with the native ligand at the same position with 0 Å RMSD, indicating the reliability of the docking protocol (Fig. 13).
The binding affinity and interaction modes of the compounds with the target enzyme are summarized in Table 5.The results showed that all the compounds were able to effectively bind to the active site of CDK4 and displayed good binding energy ranging from -9.8 to 8.7, except compound 11, (− 7.7 kcal/mol) compared to the native ligand of − 11.3 kcal/mol (Table 5, Figs.14, 15).
Concerning the new active molecules, compound 6a showed the best docking score value of -9.8 kcal/mol and stabilized in the active pocket through thirteen hydrophobic interactions with amino acid of CDK4 active site Ile12, Gln98, Asp99, Phe9, Val20, Val72, Ala33, Phe93, Lys35, Ala157, Ile12, and Leu147 residues, respectively (Table 5, Fig. 15A).Whereases, the existence of bromine has had a positive impact on the binding of compound 6b to the enzyme active pocket.This can be seen through the formation of two hydrogen bonds with the amino acid Asp99, Val96 and thirteen hydrophobic interactions with Leu147, Ile12, Ala157, Lys35, Phe93, Ala33, Val20, and Val72 (Fig. 15B).
On the other hand, the presence of ester and two nitro groups in compound 14b enhanced the binding interaction with the CDK4 active site which formed two H-bonds (strongest interaction) with Lys22, Asp99 residues as well as established carbon H-bond, Pi-sigma Alkyl and Pi-Alkyl and van der Waals interactions with Glu144, Phe93, Tyr17, Val96, Ile 12 Val20, Ala33, Leu147, Lys35, Ala157 residues (Fig. 15C).Compound 14c with ester and trichloro phenyl moieties demonstrated one H-bond and eleven hydrophobic interactions (Fig. 15D).
Furthermore, the 4-oxothiazolidine ring in compound 17 played a key role in its stabilization in the active pocket, forming two H-bonds with Asn145, Asp158 and a Pi-Sulfur interaction with Tyr17.Additionally, the carbonyl group of chromone moieties formed an H-bond with the catalytic amino acid Lys35, as well as the ester and chromone moieties established twelve hydrophobic interactions (Fig. 15E).
Finally, compound 19 revealed a good binding energy of − 9.1 kcal/mol and formed a stable complex with the active pocket through the formation of three bonds, one carbon H bond and seven hydrophobic interactions (Fig. 15F).
Based on the bioavailability radar chart, it was observed that compound 14c falls within the optimal range (pink area) for the six major variables, namely lipophilicity, size, polarity, solubility, saturation, and flexibility.This result indicates a good likelihood of oral bioavailability for this compound.Conversely, compounds 6a and 6b are not expected to be orally bioavailable as they were far from the ideal saturation range.Moreover, compounds 11, 14b, 17 and 19 were found to be slightly outside the ideal polarity and flexibility ranges (Fig. 16).
According to the predicted data, the tested compounds were found to be absorbed in the human intestine, water-soluble and showed oral bioavailability except for compounds 11 and 19.Only compounds 6a and 6b were predicted to have Caco2 permeability.Moreover, all hits had a positive effect on P-gp inhibitors and could break the blood-brain barrier, which could be a promising avenue for future research in exploring bioactive molecules targeting nervous system diseases except compound 9 (Table 7).The molecule's ability to inhibit or substrate cytochrome P450 (CYP450) served as a representative of the metabolism criteria.Some isoforms are responsible for 90% of the oxidative stress, while substrates of CYP2C9, 2D6, 3A4, and inhibitors of CYP1A2, 2D6, 2C9, 2C19, 3A4 indicate the likelihood of Drug-drug interaction phenomenon with other drugs.In our records, the tested compounds displayed inhibition effects on some CYP450 isoforms 35 (Table 7).
The OCT2 (organic cation transporter 2) is the first step in the renal secretion of many cationic drugs, and its inhibitors may alter the way drugs accumulate in the kidney and cause nephrotoxicity 36 .None of the tested compounds inhibited OCT2.Ultimately, the anticipated toxicity profile of the investigated compounds showed that the majority of them had negative skin sensitization and no Ames negative hepatotoxicity (Table 7).

Conclusion
Twenty new chromone derivatives have been synthesized and their cytotoxic activity was tested against human breast cancer (MCF-7), colon (HCT-116) and liver cancer cell lines (HepG2) as well as normal skin fibroblast cells (BJ1).The result displayed that Compounds 14b, 17, and 19 showed cytotoxic activity against MCF-7, whereas compounds 6a, 6b, 11 and 14c exhibited highly potent activity toward HCT-116 cancer cell lines.The potential Table 6.Physicochemical properties of the most active synthesized compounds 6a, 6b, 11, 14b, 14c 17 and 19 using SwissADME online server.MW molecular weight, Log P lipophilicity (log octanol/water partition coefficient), HBA hydrogen bond acceptor, HBD hydrogen bond donor, MR molar reactivity, TPSA topological polar surface area.Drug likeness (Lipinski Pfizer filter) limits are "Yes, drug-like" for MW ≤ 500, Log p (MLOGP) ≤ 4.15, HBA ≤ 10, and HDD ≤ 5. Veber GSK filter for nRB ≤ 10, TPSA ≤ 140 Å 2 .www.nature.com/scientificreports/cytotoxic effects of these compounds may be due to their ability to induce DNA fragmentation in cancer cell lines, down-regulate the expression level of CDK4 as well as the anti-apoptotic gene Bcl-2 and up-regulate the expression of the pro-apoptotic genes P53 and Bax.Additionally, compounds 14b and 14c showed a dual mechanism of action via apoptosis and cell cycle arrest induction.The molecular docking study was carried out to understand the binding interaction of the most active synthesized compounds with the binding sites of CDK4.Compounds 6a, 14b, 17, and 19 showed good binding energy and formed stable complexes with the enzyme active pocket.Moreover, the bioavailability radar chart, showed that compound 14c falls within the optimal range for the six major variables, namely lipophilicity, size, polarity, solubility, saturation, and flexibility.

General information
All reagents and solvents were of commercial grade.Visnagin (Sigma-Aldrich ChemieGmeH,Taufkirchen, Germany).Melting points were determined on the digital melting point apparatus (Electro thermal 9100, Electro thermal Engineering Ltd., serial No. 8694, Rochford, United Kingdom) and are uncorrected.The reaction progress was monitored by thin-layer chromatography (TLC) using silica gel plates (POLYGRAM SILG/UV254, 0.20 mm), which were visualized under UV light 254 and 365 nm.The IR spectra were detected utilizing the Brukur-5000 FTIR spectrometer.The 1 H and 13 C NMR spectra were recorded using a JEOL-ECA-50 NMR instrument at 500 and 125 MHz, respectively, using TMS as the internal standard, National Research Center, Egypt.Hydrogen coupling patterns are described as (s) singlet, (d) doublet, (t) triplet, (q) quartetand (m) multiple.Chemical shifts were defined as parts per million (ppm) relative to the solvent peak.Mass spectra (EI) were identified on Finnegan MatSSQ 7000 mode: EI, 70Ev (Thermo Inst.Sys. Inc., USA).6-formyl-7-hydroxy-5-methoxy-2-methyl  (8) were prepared according to the reported methods 23,24 .

Cell cycle arrest and apoptosis detection
Cellular DNA content was analyzed using a flow cytometer.After 24 h of the culture of HepG2 and treated with naringin and NDN at concentrations of 188.26 and 214.57µg/mL, respectively, DNA was stained with propidium iodide (PI) (ab139418 PI Flow Cytometry Kit/BD, Sigma, St. Louis, MO) to determine cell cycle distribution.The cell distribution percentage in the G0/G1, G2/M, and S phases was measured, and the cell cycle profile was calculated based on the results.Cells were stained with annexin V-FITC and PI labelling as directed by the manufacturer (BioVision, Annexin V-FITC Apoptosis Detection Kit, USA, Catalog #: K101-25) to determine apoptotic cell populations.A flow cytometry analysis of cell cycle distribution (FACS) was performed using a FACS.

Molecular docking
The molecular docking was performed using AutoDock Vina in PyRx software version 8 43 .The three-dimensional structure of CDK4-Cyclin D3 bound to abemaciclib was acquired from the RCSB protein data bank in the PDB format utilizing 7SJ3 code (https:// www.rcsb.org/ struc ture/ 7SJ3 access on 12 September 2023).The unwanted co-crystallized ligand and water molecules were removed and the enzyme was prepared using the QuickPrep tool module in the MOE program, saved as pdb and converted to PDBQT format by Autodock vina tools.Our docking protocol was validated by re-docking the co-crystallized ligand,abemaciclib (N-{5-[(4-ethylpiperazin-1-yl)methyl]pyridin-2-yl}-5-fluoro-4-[4-fluoro-2-methyl-1-(propan-2-yl)-1H-benzimidazol-6-yl]pyrimidin-2-amine).The chemical structure of the selected molecules was constructed with the ChemDraw ultra 10.0, saved as an SDF file then minimized by applying the MMFF94 force field and converted to a pdbqt file using OpenBable tools involved in Pyrx software.AutoDock Tools was employed to set the size and the centre of the grid box.The size of the CDK4 active site was set at 22.55 × 13.25 × 22.98 Å coordinates in x, y, and z dimensions and centred to x = 13.14, y = -38.18,z = 10.07.PyRx software presents the 9 most suitable docking poses of the ligand-protein complex after the docking is completed and subsequently ranked according to the binding energy.We have selected the first docking pose which is the most suitable pose where the ligands have the lowest binding energy, zero Å root-mean-square deviation (RMSD)and strongly interact with the protein's catalytic cavity and visualized them using BIOVIA Discovery Studio Visualizer to have a great insight into ligand binding position in the protein cavity.

Figure 7 .Figure 8 .
Figure 7.The RT-qPCR validation of mRNA expression for P53, BAX, BCL2, CDK4, in MCF7, Breast cancer cell line (mcF7) among groups of control, DOX; NC: normal control (cancer cells without any treatment); Error bars represents standard error of mean (SEM).Means comparisons were performed by using One-Way ANOVA test.*Significant differences.

Figure 9 .
Figure 9. Flow cytometry analysis using Annexin V FITC and propidium iodide (PI) for apoptosis measurements for compound 14b, 17, 19 and DOX in MCF cells.

Figure 11 .
Figure 11.Effect of compound 14b on cell cycle arrest in MCF cells.

Figure 12 .
Figure 12.Effect of compound 14c on cell cycle arrest HCT 116 Cells.

Figure 13 .
Figure 13.A The 3D conformations of the native ligand, abemaciclib (green) and re-docked ligand (brown) within the active site of CDK4-Cyclin D3 (PDB ID: 7SJ3); showed that they were superimposed in the same position.B The 2D conformations of the re-docked abemaciclib within the active site of CDK4-Cyclin D3 (PDB ID: 7SJ3).

Figure 14 .
Figure 14.The 3D conformations of the native ligand (green), re-docked ligand (brown) and all docked compounds a within the active site of CDK4-Cyclin D3 (PDB ID: 7SJ3); showed that they were superimposed in the same position.

Figure 16 .
Figure 16.Bioavailability radar chart of the potent chromone congers 6a, 6b, 11, 14b, 14c, 17 and 19.The ideal value for each oral bioavailability factor was shown in the pink region, and the expected ones for the assessed molecules were shown as red lines the colored zone is the typical physiochemical space for oral bioavailability.

Table 2 .
IC 50 of the highly anti-proliferative active compounds against human Colon and Human breast cancer cell lines.

Table 7 .
Prediction of some of the ADMET end points of the most active synthesized compounds 6a